Resonance Assignment/Principles and concepts

Introduction

This section is intended to introduce a few definitions and concepts in resonance assignment protocols. For in depth description of the process see e.g. Kurt Wütrich's book NMR of Proteins and Nucleic Acids (Wiley, 1986) and John Cavangh's et al. textbook Protein NMR Spectroscopy. Principles and Practice (2^nd edition, Elsevier, 2007). 

Stable isotope labeling schemes

Through the NESG consortium, the most prevalent isotope labeling schemes are as follows:

• 100% ¹⁵N, 100%¹³C-labeled (or doubly-labeled) samples are the main category, to which the majority of the information on this site applies. They are used for complete resonance assignments and structure calculation.
  
• 100% ¹⁵N-labeled samples are used for screening with 15N-HSQC. They can also find limited use in collecting RDC-type data.
  
• 100% ¹⁵N, 5-7% ¹³C-labeled samples are used to obtain stereospecific assignment of Val and Leu side chain methyl groups, usually important for proper packing of hydrophobic core.
• 100% 14N, 100% 12C (or unlabeled) or alternatively natural abundance samples can be used in 50%-50% mixtures for homodimer structure determination.
  

In addition to these labeling schemes, one can find it useful, especially larger proteins to have selectively labeled samples, such as SAIL NMR (http://www.sailnmr.org/). To reduce signal broadening due to spin-spin relaxation, it may be advantageous to deuterate the protein to a certain level.

NMR experiments

This section describes the types of connectivities that can be established between nuclei by given experiments. For specifics and experimental setup, please refer to the NMR Data Collection section of this site.

Through bond

• ¹⁵N-HSQC - correlates a ¹⁵N nucleus and a 1H directly attached to it. Mainly used to identify backbone amide groups.
  
• ¹³C-HSQC - correlates a ¹³C nucleus and a 1H directly attached to it. Can cover aliphatic and/or aromatic range of ¹³C chemical shifts
  
• Constant time - enhances resolusion. Peak sign gives information on the number of ¹³C neighbors a given ¹³C nucleus has
• HNCO, 3D - correlates a backbone amide with the C' of preceding residue or a C<math>/gamma</math>/C<math>\delta</math> of Asn/Gln with NH₂ of the side chain
• HNCA, 3D - correlates a backbone amide with the C<math>\alpha</math> of the same and preceding residues
• HNCACB, 3D - correlates a backbone amide with the C<math>\alpha</math> and C<math>\beta</math> of the same and preceding residues. Usually has C<math>\beta</math> and C<math>\alpha</math> peaks of opposite signs
• CBCA(CO)NH, 3D - correlates a backbone amide with the C<math>\alpha</math> and C<math>\beta</math> of the preceding residue